JP2669226B2 - Capillary electrophoresis device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a capillary zone electrophoresis apparatus for separating and analyzing charged substances such as amino acids, proteins and nucleic acids, and separation and analysis including neutral substances by interaction with components contained in a buffer solution. The present invention relates to a capillary electrophoresis apparatus that collectively refers to both types of interfacial conductivity chromatography.

[0002]

2. Description of the Related Art In a capillary electrophoresis apparatus, a substance which electrophoreses in a capillary and a neutral substance which moves by an electroosmosis method are detected by a detector by a voltage applied to both ends of the capillary, and a data processing apparatus is used based on the detection signal. Detects the elution time of each peak, and identifies the component of each peak from the elution time. Since the mobility of each peak component is determined by the type of component and the situation in which it is placed, the retention time is an index for identification as in the case of chromatography. Moving velocity Vi of each component in capillary electrophoresis
Is given by Vi = ( Mi + Me.o) E where E is the strength of the electric field, Mi is the effective mobility of the ion species i as an ion, and Me.o is the mobility of the electroosmotic flow due to the electric double layer on the inner surface of the capillary. . The mobility Me.o of the electroosmotic flow changes depending on the surface condition of the inner surface of the capillary. M
Since the ion elution time changes when eo changes, various cleaning methods such as a method using an alkaline solution, a method using water, and a method using a buffer solution have been proposed as the cleaning method in order to sufficiently clean the inner surface of the capillary. ing.

[0003]

Since the state of the inner surface of the capillary is not known during the washing of the capillary, a method is used in which the washing is performed under certain conditions and then the analysis is actually performed to confirm whether the washing is sufficient. ing. Therefore, the cleaning efficiency is poor. If the washing state of the capillaries can be known during washing, it is convenient because, for example, when the washing state reaches a prescribed state, the washing can be ended and the analysis can be started. Therefore, it is an object of the present invention to provide a capillary electrophoresis apparatus capable of efficiently performing a cleaning operation by making it possible to know the cleaning state during cleaning.

[0004]

In the present invention, the surface condition of the inner surface of the capillary is measured during liquid transfer by measuring the flow voltage generated when a pressure difference is generated between both ends of the capillary to transfer the liquid through the capillary. Know and feed it back to the cleaning operation so that the analysis can always be performed in a stable state. Therefore, in the present invention, means for feeding the liquid into the capillary by the pressure difference between both ends of the capillary,
And a means for measuring a flow voltage generated between both ends of the capillary when liquid is fed into the capillary under a state where a pressure difference is generated between both ends of the capillary.

[0005]

[Function] Flow velocity Ve.o by electroosmotic flow (= Me.o.E)
Is expressed as follows. Ve.o = εζE / 4πη where ζ is the zeta potential of the electric double layer, and ε and η are the dielectric constant and viscosity coefficient of the substance filling the electric double layer, respectively. From Ohm's law, V = IL / ak. Here, V is voltage, I is current, L is length, a is cross-sectional area, and k is specific conductivity. Therefore, Ve.o = (εζ / 4πη) (V / L) = (εζ / 4πηk) (I / a) (1)

On the other hand, a streaming potential (Stream) generated at both ends when a liquid is moved through a capillary by a phenomenon opposite to electroosmosis.
ing Potential) Vst is expressed as Vst = (εζ / 4πηk) P (2). Here, P is a pressure difference between both ends of the capillary. Therefore, if the pressure difference P between both ends of the capillary is constant, the coefficient (εζ / 4π) is changed by the fluctuation of the streaming potential Vst.
It is possible to know the fluctuation of ηk), and when P is changed, it is possible to know the fluctuation of the coefficient (εζ / 4πηk) by measuring P.

On the other hand, in the equation (1), the cross-sectional area a is considered to be almost unchanged, the current I generated by electroosmosis is I = Vak / L, the voltage V during migration is known, L is unchanged, and Although the specific conductivity k in the multilayer is not known, it is considered that k is constant if the value of (εζ / 4πηk) is constant. Therefore, by measuring the streaming potential Vst, the surface state of the inner surface of the capillary contributing to electroosmosis can be determined. I can know.

By measuring the streaming potential Vst during washing and ending the washing when it reaches a steady value, electroosmosis can be analyzed in a constant state. When the capillary is first washed with the washing liquid and then the flowing voltage is measured by replacing the liquid sending with the buffer liquid, the analysis can be started immediately when the flowing voltage reaches a steady value.

[0009]

DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 is a schematic structural view showing an entire embodiment.
FIG. 2 is a cross-sectional view showing the liquid feeding section in detail. In FIG. 1, one turntable 7 holds a plurality of vials 9, and the vials 9 contain a sample, a buffer solution, a cleaning solution, and the like. The other turntable 8 also holds a plurality of vials 10, and the vial 10 contains a buffer solution or is empty. The turntables 7 and 8 are rotatably supported on a table 16 by respective rotation shafts. A pulley 13 is attached to the lower end of the rotary shaft of the turntable 7, and a pulley 14 attached to the rotary shaft of a stepping motor 15 for driving.
The belt 12 is hung between them and the turntable 7 is driven by the stepping motor 15 to rotate. Similarly, the other turntable 8 has a pulley 13a attached to the lower end of its rotary shaft, and a pulley 14a attached to the rotary shaft of a driving stepping motor 15a.
The belt 12a is hung between the turntable 7 and
Are rotated by being driven by the stepping motor 15a.

A female screw is provided on the table 16, and the female screw is engaged with a ball screw 17 attached to a rotation shaft of a stepping motor 19 by a coupling 18. The rotation of the ball screw 17 causes the table 16 to move up and down. The two turntables 7 and 8 also move in the vertical direction with the movement. Above the turntables 7 and 8, a capillary 1 made of fused quartz is held. One end of the capillary 1 is fixedly held by the holding member 32 together with the electrode 2 arranged closely, and the other end of the capillary 1 is fixedly held by another holding member together with the electrode 3 arranged closely. In addition, a detector 4 is arranged on the other end side of the capillary 1. One end of the capillary 1 and the electrode 2 are arranged so as to be inserted into the vial 9 held by the turntable 7, and the other end of the capillary 1 and the electrode 3 are arranged in the vial 10 of the other turntable 8.
It is placed at the position to be inserted into.

In order to pressurize and deliver the cleaning liquid or the buffer liquid into the capillary 1 from one end thereof, the vial 9 is inserted when the electrode 2 and one end of the capillary 1 are inserted into the vial 9.
As shown in FIG. 2, the holding member 32 is a cap of the vial 9 so that the inside can be hermetically sealed, and the sealing member 27 for sealing the inside of the vial 9 is provided, and the capillary 1 is kept airtight by the seal 28. The electrode 2 is also held airtightly. A pipe 30 is connected through a joint 29 of a holding member 32 to pressurize the inside of the vial 9,
The pipe 30 is connected to a gas pressure source or an opening by a three-way solenoid valve 31.

Returning to FIG. 1, at one end of the capillary 1, contacts 20 and 21 are attached to a rack 22 in order to contact the high-voltage contact 20 or the flowing voltage measurement contact 21 with the electrode 2. Reference numeral 22 meshes with a gear 23 attached to a rotating shaft of a motor 24. Capillary 1
The electrode 3 on the other end side is connected to a switching relay 6, and the relay 6 is connected to the high voltage power source 5 and the voltage measuring device 25 by switching. The high voltage power supply 5 has an anode connected to the high voltage contact 20, a cathode grounded, and a relay 6 connected to the electrode 3. The streaming voltage measuring contact 21 is connected to a voltage measuring device 25 and the voltage measuring device 2
5 is connected to the electrode 3 via the relay 6. A controller 26 controls the rotation of the turntables 7 and 8 and the vertical movement of the table 16, and also controls the on / off operation of the high-voltage power supply 5 and the measurement operation of the voltage measuring device 25.

Next, the operation of this embodiment will be described.
By rotating the turntable 7 to select an arbitrary vial 9 and raising the table 16, the selected vial 9 is sealed by the seal 27 of the holding member 32.
One end of the capillary 1 and the electrode 2 are inserted into the vial 9. At this time, the other end of the capillary 1 and the electrode 3 are inserted into the vial 10 of the turntable 8. When the capillary 1 is washed, the vial 9 containing a washing solution is selected. Electrode 2 with vial 9 sealed
The flow voltage measuring contact 21 is brought into contact with the three-way solenoid valve 3
1 is switched to the gas pressure source side, and the cleaning liquid in the vial 9 flows into the capillary 1 and is discharged to the vial 10 side. At this time, the relay 6 is switched to the voltage measuring device 25 side, the voltage between the electrodes 2 and 3 is measured by the voltage measuring device 25, the flowing voltage is measured, and the value is sent to the controller 26. The washing operation is terminated when the streaming voltage reaches a steady state.

As another cleaning method, the cleaning is started with the cleaning liquid, the table 16 is temporarily lowered during the cleaning, and the turntable 7 is turned on.
The buffer solution is selected, the table 16 is raised again, one end of the capillary 1 and the electrode 2 are inserted into the vial 9 and sealed, and the other end of the capillary 1 and the electrode 3 are inserted into the vial 10 to remove the vial 9. The buffer solution is pressurized and the buffer solution is sent into the capillary 1, and the flow voltage between both ends of the capillary is measured.
When the flowing voltage reaches a steady state, the cleaning operation is terminated,
It is also possible to switch to the measurement operation immediately.
In the embodiment, pressure is applied to feed the liquid to the capillary.
On the contrary, the vial 9 on the side of the electrode 2 may be opened, and the vial 10 on the side of the electrode 3 may be hermetically sealed and put in a depressurized state, whereby liquid can be sent to the capillary.

[0015]

According to the present invention, the cleaning state of the inner surface of the capillary is known by detecting the flowing voltage during the cleaning of the capillary, so that the cleaning state of the capillary can be known unless the sample is actually analyzed. What did not exist
According to the present invention, it becomes possible to determine the washing state in the washing process, and it is possible to always perform analysis in the same state. When cleaning is performed using a plurality of liquids, it is difficult to judge whether or not the cleaning effect is sufficiently exhibited by each of the cleaning liquids in the related art, but the present invention enables such judgment. Therefore, the cleaning process does not take an unnecessarily long time, and the cleaning can be performed in the shortest time according to the state inside the capillary.

[Brief description of the drawings]

FIG. 1 is a schematic configuration diagram showing an entire embodiment.

FIG. 2 is a cross-sectional view of a liquid delivery part in the example.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Capillary 2, 3 electrode 4 Detector 5 High voltage power supply 6 Switching relay 7, 8 Turntable 9, 10 Vials 20 High voltage contact 21 Streaming potential measurement contact 25 Voltage measuring device 31 Three-way solenoid valve

Claims (1)

(57) [Claims] 1. A detector detects a substance electrophoresing in the capillary and a neutral substance moving by an electroosmosis method by a voltage applied to both ends of the capillary, and the elution time of each peak is measured from the detection signal. In a capillary electrophoresis apparatus, a means for sending liquid into the capillary by a pressure difference between both ends of the capillary, and a flow voltage generated between both ends of the capillary by sending the liquid into the capillary in a state where a pressure difference is generated between both ends of the capillary. A capillary electrophoresis apparatus comprising: